1) Field of the Invention
The present invention relates to a confocal scanning microscope that irradiates a fluorescent-dyed specimen with stimulating light and detect fluorescence emitted from the specimen for observation of spatial fluorescence distribution and influence of the stimulating light.
2) Description of the Related Art
It is known that confocal scanning microscopes that employ a stimulating laser to stimulate a specimen are effectively used in the caged method, Fluorescence Loss in Photobleaching (FLIP), or the like. According to the caged method, a caged reagent and a fluorescence indicator, which is calcium sensitive to indicate the calcium ion concentration, are injected into a specimen, and a stimulating laser beam is irradiated to a portion of the specimen to generate a cleavage in a caged group of the caged reagent. When the caged group is cleaved, substances contained inside is discharged and the calcium ion concentration thereof is changed. An exciting laser beam is irradiated onto the specimen for the fluorescent observation of the change in calcium ion concentration over time. On the other hand, according to the FLIP, a specific portion in a cell is bleached through repetitive irradiation with a stimulating laser beam. Then, diffused protein causes decrease in fluorescence intensity in a surrounding area. The process of decreasing fluorescence intensity is observed over time with the use of an exciting laser beam, which allows the observation of fluorescence emitted from the irradiated specimen. In both the caged method and the FLIP, during fluorescence observation of the specimen over time, it is important to maintain two different irradiations of laser beams to the specimen, that are, the stimulating laser beam which gives light stimulus to a specific portion of the specimen and the exciting laser beam which allows the fluorescence observation (see U.S. Pat. No. 6,094,300).
Recently, there is a high demand for the realization of three-dimensional fluorescence observation of the specimen. Also for the caged method and the FLIP mentioned above, it is highly demanded that three-dimensional fluorescence observation be realized through the irradiation of an exciting laser beam for observation achieved while the light stimulus is given to an optional portion through the irradiation of a stimulating laser beam, for example. However, the light collective plane perpendicular to the optical axis of the stimulating laser beam and the light collective plane perpendicular to the optical axis of the exciting laser beam for observation is determined according to the distance between the specimen and the objective lens, and thus basically they are the same plane. Therefore, in the three-dimensional fluorescence observation of the specimen, if the distance between the specimen and the objective lens is changed by a microscope focus adjusting mechanism such as a stepping motor, the light collective plane of the stimulating laser beam and the light collective plane of the exciting laser beam for observation are both changed so that the light stimulus cannot be provided continuously to a desired area in the three-dimensional space in the specimen. Thus, it may be difficult to observe the fluorescent specimen three-dimensionally while providing the light stimulus continuously or repeatedly by the stimulating laser beam to an area including an optional point in the specimen. Hence, there may be a problem that the advantage of a confocal scanning microscope that the three-dimensional fluorescence observation is possible by the confocal effect in combination with a light stimulus, can not be exerted, thus a fluorescent image cannot be obtained in a desired condition.